title: Flow sorting and sequencing meadow fescue chromosome 4F.
abstract: The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.
authors.affiliation: Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, 78371 Olomouc, Czech Republic.
authors.forename: David
authors.initials: D
authors.lastname: Kopecký
authors.forename: Mihaela
authors.initials: M
authors.lastname: Martis
authors.forename: Jarmila
authors.initials: J
authors.lastname: Číhalíková
authors.forename: Eva
authors.initials: E
authors.lastname: Hřibová
authors.forename: Jan
authors.initials: J
authors.lastname: Vrána
authors.forename: Jan
authors.initials: J
authors.lastname: Bartoš
authors.forename: Jitka
authors.initials: J
authors.lastname: Kopecká
authors.forename: Federica
authors.initials: F
authors.lastname: Cattonaro
authors.forename: Štěpán
authors.initials: Š
authors.lastname: Stočes
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.forename: Pavel
authors.initials: P
authors.lastname: Neumann
authors.forename: Jiří
authors.initials: J
authors.lastname: Macas
authors.forename: Hana
authors.initials: H
authors.lastname: Šimková
authors.forename: Bruno
authors.initials: B
authors.lastname: Studer
authors.forename: Torben
authors.initials: T
authors.lastname: Asp
authors.forename: James H
authors.initials: JH
authors.lastname: Baird
authors.forename: Petr
authors.initials: P
authors.lastname: Navrátil
authors.forename: Miroslava
authors.initials: M
authors.lastname: Karafiátová
authors.forename: Marie
authors.initials: M
authors.lastname: Kubaláková
authors.forename: Jan
authors.initials: J
authors.lastname: Šafář
authors.forename: Klaus
authors.initials: K
authors.lastname: Mayer
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Doležel
mesh_terms: D015139:Blotting, Southern; D002874:Chromosome Mapping; D032461:Chromosomes, Plant / Q000235:genetics*; D031751:Festuca / Q000235:genetics*; D023061:Gene Order; D018745:Genome, Plant / Q000235:genetics; D023281:Genomics / Q000379:methods*; D001467:Hordeum / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping / Q000379:methods; D008969:Molecular Sequence Data; D012275:Oryza; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D045868:Sorghum / Q000235:genetics; D026801:Synteny